PBMCs were isolated from NHP whole blood using Ficoll gradients, according to manufacturer’s instructions. Plasma was stored at −80°C until further use. PBMCs were stained for monocytes and dendritic cells using the following antibody panel, CD16-BV605, CD14-PE-Cy7, CD11c-PE, HLA-DR-BV786, CD123-PE-CF594, CD20-BV711, CD159a-APC, Live/Dead-APC-Cy7 (BDbiosciences). Following a 30 min incubation with the antibody master mix, PBMCs were washed with staining buffer [1x PBS (Invitrogen), 2% FCS (Hyclone), 2 mM EDTA (Invitrogen), 15 mM HEPES (Invitrogen)] and analysed on BD LSR II.
Cd16 bv605
Manufactured by BD
Sourced in United States
The CD16-BV605 is a fluorescently-labeled antibody used for the detection and analysis of the CD16 cell surface marker. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Hla dr bv786, by BD (2 mentions)
F4 80 pe, by Thermo Fisher Scientific (2 mentions)
Cd11b apc cy7, by BD (2 mentions)
Cd11b fitc, by BD (2 mentions)
Sca 1 fitc, by BioLegend (2 mentions)
Ckit alexa fluor 700, by Thermo Fisher Scientific (2 mentions)
Cd14 pe cy7, by BD (1 mentions)
Cd11c pe, by BD (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Cd20 bv711, by BD (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
Cd14 apc cy7, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Live dead fixable violet dead stain kit, by Thermo Fisher Scientific (1 mentions)
Cd56 apc, by BD (1 mentions)
Cd19 bv510, by BD (1 mentions)
Cd3 buv395, by BD (1 mentions)
Easysep human nk cell enrichment kit, by STEMCELL (1 mentions)
6 protocols using cd16 bv605
1
Multicolor Flow Cytometry of NHP PBMCs
2
Multiparametric Immune Cell Profiling
3
Myeloid Cell Phenotyping with DCFDA
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The viably cryopreserved PBMC were thawed and stained with LIVE/DEAD fixable violet Dead Stain Kit (Thermo Fisher Scientific) for 20 minutes followed by staining with a myeloid panel of antibodies comprising CD14-APC-Cy7 (564123), CD33-PE-Cy7 (333952), CD16-BV605 (563172), HLA-DR-BV786 (564041), all from BD Biosciences, for 30 minutes at 4°C. Cells were then washed and stained with H2-DCFDA (D399, Molecular Probes) in serum-free Iscoves’ Modified Dulbecco's Medium (IMDM; 36531, Thermo Fisher Scientific) for 30 minutes at 37°C, washed and acquired on a five laser BD LSR Fortessa and analyzed with FlowJo.
4
Multiparametric Immune Cell Profiling
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Macrophages were stained with CD11b-FITC (BD 561688), F4/80-PE (eBioscience 12-4801-80) with anti-Mouse TCR Vα 11.1/11.2-FITC and Vα2-PE as isotype controls. CMPs and GMPs were analysed by Sca-1-FITC (Biolegend 122505), cKit-Alexa Fluor 700 (eBioscience 56-1172-80), CD11b-APC-Cy7 (BD 557657) and CD16-BV605 (BD 563006).
5
NK Cell Enrichment and Cytokine Stimulation
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Cryopreserved immune cells were thawed and enriched for NK cells using the EasySep™ Human NK Cell Enrichment Kit (STEMCELL Technologies, Cambridge, MA). Enriched NK cells were stained with the following surface markers for flow cytometry: Live/Dead Blue, CD56 APC, CD3 BUV395, CD19 BV510 (BD, Franklin Lakes, NJ), CD18 APC-Cy7, CD7 BV711, CD16 BV605, and IFITM1 FITC. Cells were then stimulated with the following for 15 min: 500U/ml IL-2, 50ng/ml IL-12, 500ng/ml LPS, 500U/ml IFNαa4, and 1ug/ml α-mouse IgG. After cytokine stimulation, cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized with methanol for 30 min. Samples were then stained with the following intracellular antibodies overnight: pRPS6 BV421 (Biolegend, San Diego, CA). Samples were ran on a 5-laser Aurora (Cytek Biosciences, Fremont, CA).
6
Multiparameter Flow Cytometry Analysis
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Multiparameter FACS analysis was performed using a monoclonal antibody (mAb) panel to measure activation in blood [18] . The following mAb combinations, relevant for this manuscript, were used: CD45BUV496 (Becton & Dickinson, Franklin Lakes, NJ, USA (BD), cat 741185), HLA-DRBUV805 (BD, cat 748338), CD20V450 (BD, cat 655872), CD16BV605 (BD, cat 563916), CD3BV650 (BD, 563916), CD40BV750 (BD, cat 746948), CD14PE-TxRed (Beckman Coulter, cat B92391), CD123PerCP-Cy5.5 (BD cat 558714), CD86Alexa647 (Biolegend, San Diego, CA, USA, cat 305416), CD1cAPC (Ebioscience cat 17-0015-42). Acquisition was performed on an Aurora flow cytometer (Cytek, Fremont, Ca, USA) and analyzed using FlowJo software. For analysis, the CD45 positive cells with a low side scatter (SSC) profile were first gated and then singlets were selected using a forward scatter (FSC) height versus area plot. Then, HLA-DR positive/CD3, CD20 and CD14 negative cells were selected and CD123 was plotted against CD1c to identify the plasmacytoid DC (pDC)(CD123 positive/CD1c negative) and classical DC2 (cDC2)(CD123 negative/CD1c positive) populations. Expression of CD40 and CD86 was quantified as the mean geometric mean of the fluorescence intensity (MFI).
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